Kinetics of arsenic methylation by freshly isolated B6C3F1 mouse hepatocytes

The toxic and carcinogenic effects of arsenic may be mediated by both inorganic and methylated arsenic species. The methylation of arsenicIII is thought to take place via sequential oxidative methylation and reduction steps to form monomethylarsenic (MMA) and dimethylarsenic (DMA) species, but recent evidence indicates that glutathione complexes of arsenicIII can be methylated without oxidation. The kinetics of arsenic methylation were determined in freshly isolated hepatocytes from male B6C3F1 mice. Hepatocytes (>90% viability) were isolated by collagenase perfusion and suspended in Williams' Medium E with various concentrations of arsenicIII (sodium m-arsenite). Aliquots of the lysed cell suspension were analyzed for arsenic species by hydride generation-atomic absorption spectrometry. The formation of MMAIII from sodium arsenite (1 μM) was linear with respect to time for >90 min. DMAIII formation did not become significant until 60 min. MMAV and DMAV were not consistently observed in the incubations. These results suggest that the glutathione complex mechanism of methylation plays an important role in arsenic biotransformation in mouse hepatocytes. Metabolism of arsenicV was not observed in mouse hepatocytes, consistent with inhibition of arsenicV active cellular uptake by phosphate in the medium. The formation of MMAIII increased with increasing arsenicIII concentrations up to approximately 2 μM and declined thereafter. The concentration dependence is consistent with a saturable methylation reaction accompanied by uncompetitive substrate inhibition of the reaction by arsenicIII. Kinetic analysis of the data suggested an apparent KM of approximately 3.6 μM arsenicIII, an apparent Vmax of approximately 38.9 μg MMAIII formed/L/h/million cells, and an apparent KI of approximately 1.3 μM arsenicIII. The results of this study can be used in the physiologically based pharmacokinetic model for arsenic disposition in mice to predict the concentration of MMAIII in liver and other tissues.